P53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. In 50% of human tumors, the negative regulatory activities of p53 are inactivated by gene mutation that produces a defective gene product. However, the other 50% of human tumors contain normal p53 genes with a gene product that is probably inactivated by unknown mechanisms within cancer cells. We are considering the hypothesis that normal functioning of tumor suppressor gene products in some tumors may be circumvented or functionally inactivated by differential phosphorylation. Changes in phosphorylation state of p53 can regulate its transcriptional activity, sometimes without detectable changes in p53 levels. We are studying how changes in p53 phosphorylation might affect its biological activity. We are developing methods to assess p53 phosphorylation status. Recombinant-p53 from baculovirus was used as a model to develop a mass spectrometry (MS) means for site-specific determination of phosphorylation. Okadaic acid treatment created a hyperphosphorylated species. Six phosphorylation sites and N-terminal acetylation were identified by MS. 2D PAGE was also used separate p53 phosphoisoforms and we found that each isoform was a mixture of phosphorylated species. A phosphospecific antibody has been produced to a new phosphorylation site on p53 found from this study and its properties will be examined. The MS approach for assessing in vivo p53 phosphorylation status in human tumors will be exploited by us and collaborators. In related studies, we are also examining the effects of phosphorylation upon p21-waf1, a primary effector of p53. Phosphorylation of p21-waf1 by cdk2 was shown in several wild type (wt) p53 cell lines. We are working to find the exact sites of phosphorylation and its biological implications which may relate to alteration of cdk-cyclin kinase activity. Evidence has also been generated by one of our collaborators that p53, p21-waf1 and JNK may interact directly and we will explore co-associated proteins of p21-waf1 using Proteomics methods. Altered phosphorylation of p53-dependent genes might also be an effective method of downstream p53 inactivation to escape growth control in tumor development.